Direct and continuous assay for prolyl 4-hydroxylase

Prolyl 4-hydroxylase (P4H) is a nonheme iron dioxygenase that catalyzes the posttranslational hydroxylation of (2S)-proline (Pro) residues in protocollagen strands. The resulting (2S,4R)-4-hydroxyproline (Hyp) residues are essential for the folding, secretion, and stability of the collagen triple helix. P4H uses α-ketoglutarate and O₂ as cosubstrates, and forms succinate and CO₂ as well as Hyp. Described herein is the first assay for P4H that continuously and directly detects turnover of the proline-containing substrate. This assay is based on (2S,4S)-4-fluoroproline (flp), a proline analogue that is transformed into (2S)-4-ketoproline (Kep) and inorganic fluoride by P4H. The fluoride ion, and thus turnover by P4H, is detected by a fluoride ion-selective electrode. Using this assay, steady-state kinetic parameters for the human P4H-catalyzed turnover of a flp-containing peptide were determined and found to be comparable to those obtained with a discontinuous HPLC-based assay. In addition, this assay can be used to characterize P4H variants, as demonstrated by a comparison of catalysis by D414A P4H and the wild-type enzyme. Finally, the use of the assay to identify small-molecule inhibitors of P4H was verified by an analysis of catalysis in the presence of 2,4-pyridine dicarboxylate, an analogue of α-ketoglutarate. Thus, the assay described herein could facilitate biochemical analyses of this essential enzyme.     

Monoclonal antibodies to human prolyl 4-hydroxylase

Monoclonal antibodies against human prolyl 4-hydroxylase (EC 1.14.11.2), an intracellular enzyme of collagen biosynthesis, were produced by fusing spleen cells from BALB/c mice hyperimmunized with human prolyl 4-hydroxylase and mouse myeloma cells (P3/NS 1/1-AG 4-1). Hybridomas from 14 different primary microtiter-plate well cultures produced antibodies to human prolyl 4-hydroxylase; six of them with the highest antibody titer were cloned and antibodies produced by one clone from each of the six lines were further characterized. All of the six cloned hybrids produced antibodies of the IgG class as detected by immunodiffusion. The enzyme antigen used in the present study was a tetramer composed of two pairs of different subunit proteins, alpha and beta. Only one clone which produced antibodies to the alpha subunit was obtained, the other five antibodies being directed against the beta subunit. All the antibodies reacted with the tetramer form of the enzyme. Species cross-reactivity of the antibodies was tested using cultured human, mouse and chick fibroblasts and purified prolyl 4-hydroxylase from chick and mouse sources. None of the antibodies cross-reacted with chick or mouse fibroblasts, as determined by immunofluorescence, whereas one antibody reacted with purified chick and mouse prolyl 4-hydroxylase when examined by the western blotting technique. This antibody caused a strong inhibition of human prolyl 4-hydroxylase activity, but the other five antibodies had negligible inhibitory effect on the activity of the enzyme.     

Dissociation and reassociation of oligomeric viral glycoprotein subunits in the endoplasmic reticulum.

The vesicular stomatitis virus (VSV) glycoprotein (G) forms noncovalently linked trimers in the endoplasmic reticulum (ER) prior to transport to the cell surface. Here we examined the formation of heterotrimers between wild-type and mutant subunits that were retained in the ER by C-terminal retention signals. When G protein was coexpressed with mutant subunits that formed trimers at the wild-type rate and were transported from the ER at the wild-type rate, heterotrimers were readily detected. In contrast, when G protein was coexpressed with mutant subunits that formed trimers at the wild-type rate, but were retained in the ER, heterotrimers were not detected unless transport of the wild-type molecules from the ER was blocked. After removal of transport block, the heterotrimers then dissociated and reassorted to homotrimers of the mutant protein that were retained in the ER and wild-type trimers that were transported to the cell surface. These and other results presented here indicate that there is an equilibrium between G protein trimers and monomers in vivo, at least in the ER. This equilibrium may function to allow escape of wild-type subunits from aberrant retained subunits.     

Inhibition of prolyl 4-hydroxylase by hydroxyanthraquinones.

Prolyl 4-hydroxylase (EC 1.14.11.2) is an essential enzyme in the post-translational modification of collagen. Inhibitors of this enzyme are of potential interest for the treatment of diseases involving excessive deposition of collagen. We have found that anthraquinones with at least two hydroxy groups ortho to each other are potent inhibitors of this enzyme. Kinetic studies revealed that 2,7,8-trihydroxyanthraquinone (THA) competitively inhibited the co-substrate, 2-oxoglutarate, but was non-competitive with regard to ascorbate and was tentatively considered to be uncompetitive with regard to protocollagen. The inhibition by THA was greatly enhanced in the absence of added Fe2+ and was partially reversed by the addition of concentrations of Fe2+ in excess of the optimum for the enzymic reaction. Binding studies indicated that THA is an effective chelating agent for Fe2+. Several non-quinoidal compounds bearing the catechol moiety also inhibited the enzyme. The results suggest that THA inhibited prolyl 4-hydroxylase by binding to the enzyme at the site for 2-oxoglutarate possibly involving the Fe2+ atom, rather than by complexing with Fe2+ in free solution. The inhibition of prolyl 4-hydroxylase by THA exhibited strong positive co-operativity and may involve three distinct but non-independent binding sites.     

Bioavailable affinity label for collagen prolyl 4-hydroxylase

Collagen is the most abundant protein in animals. Its prevalent 4-hydroxyproline residues contribute greatly to its conformational stability. The hydroxyl groups arise from a post-translational modification catalyzed by the nonheme iron-dependent enzyme, collagen prolyl 4-hydroxylase (P4H). Here, we report that 4-oxo-5,6-epoxyhexanoate, a mimic of the α-ketoglutarate co-substrate, inactivates human P4H. The inactivation installs a ketone functionality in P4H, providing a handle for proteomic experiments. Caenorhabditis elegans exposed to the esterified epoxy ketone displays the phenotype of a worm lacking P4H. Thus, this affinity label can be used to mediate collagen stability in an animal, as is desirable in the treatment of a variety of fibrotic diseases.     

Conformational preferences of substrates for human prolyl 4-hydroxylase

Prolyl 4-hydroxylase (P4H) catalyzes the posttranslational hydroxylation of (2 S)-proline (Pro) residues in procollagen strands. The resulting (2 S,4 R)-4-hydroxyproline (Hyp) residues are essential for the folding, secretion, and stability of the collagen triple helix. Even though its product (Hyp) differs from its substrate (Pro) by only a single oxygen atom, no product inhibition has been observed for P4H. Here, we examine the basis for the binding and turnover of substrates by human P4H. Synthetic peptides containing (2 S,4 R)-4-fluoroproline (Flp), (2 S,4 S)-4-fluoroproline (flp), (2 S)-4-ketoproline (Kep), (2 S)-4-thiaproline (Thp), and 3,5-methanoproline (Mtp) were evaluated as substrates for P4H. Peptides containing Pro, flp, and Thp were found to be excellent substrates for P4H, forming Hyp, Kep, and (2 S,4 R)-thiaoxoproline, respectively. Thus, P4H is tolerant to some substitutions on C-4 of the pyrrolidine ring. In contrast, peptides containing Flp, Kep, or Mtp did not even bind to the active site of P4H. Each proline analogue that does bind to P4H is also a substrate, indicating that discrimination occurs at the level of binding rather than turnover. As the iron(IV)-oxo species that forms in the active site of P4H is highly reactive, P4H has an imperative for forming a snug complex with its substrate and appears to do so. Most notably, those proline analogues with a greater preference for a C (gamma)- endo pucker and cis peptide bond were the ones recognized by P4H. As Hyp has a strong preference for C (gamma)- exo pucker and trans peptide bond, P4H appears to discriminate against the conformation of proline residues in a manner that diminishes product inhibition during collagen biosynthesis.     

Production of human prolyl 4-hydroxylase in Escherichia coli

Prolyl 4-hydroxylase (P4H) catalyzes the post-translational hydroxylation of proline residues in collagen strands. The enzyme is an alpha2beta2 tetramer in which the alpha subunits contain the catalytic active sites and the beta subunits (protein disulfide isomerase) maintain the alpha subunits in a soluble and active conformation. Heterologous production of the native alpha2beta2 tetramer is challenging and had not been reported previously in a prokaryotic system. Here, we describe the production of active human P4H tetramer in Escherichia coli from a single bicistronic vector. P4H production requires the relatively oxidizing cytosol of Origami B(DE3) cells. Induction of the wild-type alpha(I) cDNA in these cells leads to the production of a truncated alpha subunit (residues 235-534), which assembles with the beta subunit. This truncated P4H is an active enzyme, but has a high Km value for long substrates. Replacing the Met235 codon with one for leucine removes an alternative start codon and enables production of full-length alpha subunit and assembly of the native alpha2beta2 tetramer in E. coli cells to yield 2 mg of purified P4H per liter of culture (0.2 mg/g of cell paste). We also report a direct, automated assay of proline hydroxylation using high-performance liquid chromatography. We anticipate that these advances will facilitate structure-function analyses of P4H.     

Syncatalytic inactivation of prolyl 4-hydroxylase by anthracyclines.

The anthracyclines doxorubicin and daunorubicin were found to act as irreversible inhibitors of prolyl 4-hydroxylase. The reaction rate for enzyme from both chick and human origin was first order, the concentration of inhibitor giving 50% inhibition being 60 microM for both compounds after 1 h. The effect was dependent on the presence of iron ions in the reaction mixture. Inactivation could be prevented by addition of high concentrations of ascorbate, but not 2-oxoglutarate, before the inactivation period. The same results were obtained with competitive analogues of these cosubstrates. Lysyl hydroxylase from chick embryos was also susceptible to inactivation. Its activity was decreased by 50% after incubation for 1 h with a 150 microM concentration of the inhibitors. When chick-embryo prolyl 4-hydroxylase was incubated with [14-14C]doxorubicin, both enzyme subunits were radioactively labelled, about 70% of the total radioactivity being found in the alpha-subunit. Since the anthracyclines are known to undergo a redox reaction generating semiquinone radicals with Fe3+ only, the results suggest that the enzyme-bound iron ion is oxidized to a tervalent intermediate in uncoupled reaction cycles. The data also suggest that both enzyme subunits contribute to the catalytic site of prolyl 4-hydroxylase.     

Separation of prolyl 3-hydroxylase and 4-hydroxylase activities and the 4-hydroxyproline requirement for synthesis of 3-hydroxyproline

Differences between prolyl 3-hydroxylase and prolyl 4-hydroxylase activities were found in their stimulation and inactivation by dithiothreitol and in their affinity to poly-L-proline linked to agarose. The two enzyme activities were separated by gel filtration, the results demonstrating that they are due to separate proteins. Comparison of [¹⁴C]proline-labelled protocollagen and the same protein when fully 4-hydroxylated as substrates indicated dependence of 3-hydroxyproline formation on the presence of 4-hydroxyproline. It is suggested that the main substrate sequence for 3-hydroxyproline synthesis is -Gly-Pro-4Hyp-Gly-.     

Regulation of proline 3-hydroxylation and prolyl 3-hydroxylase and 4-hydroxylase activities in transformed cells.

Prolyl 3-hydroxylase activity and the extent of collagen proline 3-hydroxylation were studied in six transformed and three control human cell lines. In the transformed cell lines, the enzyme activity was markedly high in two, similar to that in control cells in two and significantly low in two. The extent of proline 3-hydroxylation was markedly high in cell lines with high enzyme activity, but it was also significantly high in some transformed cell lines with enzyme activities similar to those in the controls. The results thus suggest that, in addition to the amount of enzyme activity present, the rate of collagen synthesis also affects the extent of proline 3-hydroxylation in the newly synthesized collagen. The effect of acute cell transformation on prolyl 3-hydroxylase and 4-hydroxylase activities was studied by infecting chick-embryo fibroblasts with Rous sarcoma virus mutant NY68, temperature-sensitive for transformation. At the permissive temperature prolyl 3-hydroxylase activity showed a more rapid increase and decrease than did prolyl 4-hydroxylase activity, the maximal activity for both enzymes being about 2.5 times that in the control chick fibroblasts. When the transformed cells were shifted to the non-permissive temperature the decays in the elevated enzyme activities were similar, suggesting identical half-lives.     

Immunological properties of monoclonal antibodies to human and rat prolyl 4-hydroxylase

Monoclonal antibodies to human (8 clones) and rat (12 clones) prolyl 4-hydroxylase [EC 1.14.11.2] were prepared and characterized as regards subclass, subunit specificity, inhibition and crossreactivity. Among the antibodies to the human enzyme, four clones showed the IgG1 subclass, two IgA, one IgG2b, and one IgM. Four clones reacted with the alpha subunit of the enzyme, while the others reacted with the beta subunit. The enzymatic activity was inhibited by four clones. Five clones crossreacted with the rat enzyme. One clone inhibited the rat enzyme. Among the antibodies to the rat enzyme, seven clones showed the IgG1 subclass, four IgG2a and one IgG2b. Seven clones reacted with the alpha subunit, and four with the beta subunit. One reacted with neither subunit. The enzymatic activity was inhibited by seven clones. Seven clones crossreacted with the human enzyme. Three clones inhibited the human enzyme.     

The function of ascorbate with respect to prolyl 4-hydroxylase activity

1. Incubation in the presence of 2-oxoglutarate and oxygen inactivates prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase, EC 1.14.11.2), with a t 1/2 of 80 s at 37 degrees C. This inactivation is not affected by the presence or absence of the prolyl peptide substrate or added Fe(II). 2. This inactivation can be prevented by either ascorbate or dithiothreitol. It can be reversed by dithiothreitol but not by ascorbate. 3. Although the iron-containing form of prolyl 4-hydroxylase requires ascorbate for activity, ascorbate is not stoicheiometrically consumed in the reaction catalysed by the enzyme. Ascorbate cannot be replaced by alloxan, lactate, NADH plus phenazine methosulphate, dithiothreitol or L-cysteine. 4. Ascorbate has a double function with respect to prolyl 4-hydroxylase activity. On the one hand, it is required to initiate the reaction when the enzyme has become oxidized during isolation. On the other hand it is required for the protection against inactivation induced by 2-oxoglutarate and oxygen, presumably by preventing S-S bridge formation. The latter function may be of physiological importance.     

Cell density regulates prolyl 4-hydroxylase activity independent of mRNA levels

In embryonic avian tendon, cell density regulates collagen production. This control is propagated through the alpha-subunit of prolyl 4-hydroxylase where protein levels were previously shown to rise fivefold with increasing cell density. In contrast, mRNA levels are now shown not to change by both Northern and RNAse protection assays. This lack of increase contrasts with previous reports as does the mRNA length: this is 50% larger as confirmed by sequencing the 3' end. Alternative sites for cell density regulation of the enzyme could rely on its sensitivity to sulfhydryl groups. Using a fluorescent sulfhydryl probe as well as a sulfhydryl inhibitor, one observes a strong cell density response, supporting the hypothesis that cellular redox potential could alter protein stability.     

Sandwich ELISA for quantitative detection of human collagen prolyl 4-hydroxylase

Background  

We describe a method for specific, quantitative and quick detection of human collagen prolyl 4-hydroxylase (C-P4H), the key enzyme for collagen prolyl-4 hydroxylation, in crude samples based on a sandwich ELISA principle. The method is relevant to active C-P4H level monitoring during recombinant C-P4H and collagen production in different expression systems. The assay proves to be specific for the active C-P4H α₂β₂ tetramer due to the use of antibodies against its both subunits. Thus in keeping with the method C-P4H is captured by coupled to an anti-α subunit antibody magnetic beads and an anti-β subunit antibody binds to the PDI/β subunit of the protein. Then the following holoenzyme detection is accomplished by a goat anti-rabbit IgG labeled with alkaline phosphatase which AP catalyzes the reaction of a substrate transformation with fluorescent signal generation.     

Dissociation and reassociation of rabbit skeletal muscle myosin

Whereas dissociation of rabbit skeletal muscle myosin light chains occurs at an increased temperature (25°) and in the obsence of divalent cations, reassociation of the myosin oligomer requires a low temperature (4°C) and the presence of divalent cations, thus resulting in the original light to heavy chain stoichiometry. With a 5–10 per cent release of alkali light chains, LC₁ and LC₃, and a 50 per cent dissociation of the Ca²⁺ binding light chain, LC₂, there is no significant decrease in myosin ATPase activity irrespective of the cation activator, however, there is an approximate 15–20 per cent decrease in actomyosin ATPase activity. With reassociation of the myosin oligomer, actomyosin ATPase activity is partially restored as well as the original number of Ca²⁺ binding sites.